The malfunction of somatosensory circuits has been associated with the development of neuropathic pain after peripheral or central nerve injury. Microglia are the resident immune cells in the central nervous system (CNS). After peripheral nerve injury, spinal microglia facilitate the development of neuropathic pain by transforming from resting to reactive states. However, the role of brain microglia in the pathogenesis of neuropathic pain remains unexplored. We have previously shown that peripheral nerve injury results in the hyperactivity of pyramidal neurons in the mouse primary somatosensory cortex. Our preliminary studies indicate that conditional removal of brain-derived neurotrophic factor (BDNF) from microglia in the CNS prevents pyramidal neuron hyperactivity and reduces mechanical allodynia. Moreover, somatosensory cortex-targeted depletion of microglial BDNF largely recapitulates the effects of systemic BDNF depletion in neuropathic pain. Based on these findings, we hypothesize that microglia residing in the somatosensory cortex participate in the development of peripheral neuropathic pain. In this application, we will test this hypothesis by combining in vivo imaging of synapse structure and neuronal activity, gene manipulation and behavioral testing in the same animals. The proposal has two specific aims. In the first aim, we will examine the role of microglia in synaptic structural and functional alterations in the primary somatosensory cortex after peripheral nerve injury. This will test the hypothesis that microglia contribute to cortical circuit reorganization and pain hypersensitivity through BDNF-dependent mechanisms. In the second aim, we will perform cortex-targeted gene manipulation to determine whether cortical microglia directly participate in neuropathic pain through altering the cortical circuits for sensation. The proposed project explores, for the first time, the role of cortical microglia in neuropathic pain at the level of individual neurons and synapses in the living brain. Successful completion of the project will provide novel insight into the pivotal role of cortical microglia in neural circuit remodeling after peripheral nerve injury and highlight BDNF signaling in cortical microglia as an important therapeutic target for the treatment of neuropathic pain.